MANIPULATION OF AN ALTERNATIVE RESPIRATORY PATHWAY IN PHOTO-AUTOTROPHS

• US 20110059495A1
• Filed: 07/20/2010
• Published: 03/10/2011
• Est. Priority Date: 07/20/2009
• Status: Active Grant

• Current Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Original Assignee: Aurora Algae, Incorporated (Reliance Industries Limited)
• Inventors: VICK, BERTRAND, MOSELEY, JEFF, BAILEY, SHAUN
• Granted Patent: US 8,709,765 B2
• Time in Patent Office: Days
• US Class Current: 435/134
• CPC Class Codes: C12N 1/12 Unicellular algae; Culture media therefor as new plants A01H13/00
• CPC Class Codes: C12P 7/6463 obtained from glyceride producing microorganisms, e.g. single cell oil

Abstract

Exemplary methods for increasing TAG production in an algal cell during imbalanced growth conditions are provided. Some methods comprise knocking out an AOX gene, wherein the AOX gene produces an amino acid sequence having substantial similarity to the amino acid sequence of SEQ. ID. NO. 2. In further methods, the algal cell may be of genus Nannochloropsis. The AOX gene may be replaced by a construct having a nucleotide sequence having substantial similarity to SEQ ID. NOS. 3 through 5 (inclusive), wherein each of the sequences are next to or in close proximity to one another in a linear fashion. In some methods, the AOX gene may be replaced via homologous recombination. As a result, lipid production by the selected recombinant algal cell may be increased over that produced by a wild-type algal cell.

Claims

1. A method for increasing TAG production in an algal cell during imbalanced growth conditions, the method comprising:

knocking out an AOX gene.

2. The method of claim 1, wherein the AOX gene produces an amino acid sequence having substantial similarity to the amino acid sequence of SEQ. ID. NO. 2.

3. The method of claim 1, wherein the algal cell is of genus Nannochloropsis.

4. The method of claim 1, wherein the AOX gene is replaced by the construct having a nucleotide sequence having substantial similarity to SEQ ID. NOS. 3-5.

5. The method of claim 4, wherein the AOX gene is replaced by the construct of claim 4 via homologous recombination.

6. The algal cell of claim 1, wherein the AOX gene has a nucleotide sequence with substantial similarity to SEQ ID. NO. 1.

7. The algal cell of claim 4, wherein the algal cell is grown in presence of an antibiotic in which the algal cell is resistant.

8. The algal cell of claim 4, wherein the marker gene confers resistance to zeocine.

9. The algal cell of claim 4, wherein the algal cell is of genus Nannochloropsis.

10. The algal cell of claim 4, grown under imbalanced growth conditions.

11. The algal cell of claim 10, grown under starvation conditions.

12. The algal cell of claim 4, wherein lipid production is increased over that produced by a wild-type Nannochloropsis algal cell.

13. A transformation construct, the transformation construct having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA of an algal cell, the transformation construct having a second sequence of DNA similar to a corresponding second sequence of nuclear DNA of the algal cell, and the transformation construct having a sequence of DNA of interest inserted between the first and second sequences of the transformation construct.

14. The transformation construct of claim 13, wherein the DNA of interest comprises a sequence having substantial similarity to SEQ. ID. NO. 3.

15. The transformation construct of claim 13, wherein the DNA of interest comprises a sequence having substantial similarity to SEQ. ID. NO. 4.

16. The transformation construct of claim 13, wherein the DNA of interest comprises a sequence having substantial similarity to SEQ. ID. NO. 5.

17. The transformation construct of claim 14, wherein the DNA of interest further comprises a sequence having substantial similarity to SEQ. ID. NO. 4.

18. The transformation construct of claim 17, wherein the DNA of interest comprises a sequence having substantial similarity to SEQ. ID. NO. 5.

19. A transformation method for introducing deoxyribonucleic acid (DNA) into the nucleus of an algal cell, the method comprising:

preparing a transformation construct, the transformation construct having a first sequence of DNA similar to a corresponding first sequence of nuclear DNA, the transformation construct having a second sequence of DNA similar to a corresponding second sequence of the nuclear DNA, the transformation construct having a sequence of DNA of interest inserted between the first and second sequences of DNA of the transformation construct, and

transforming a target sequence of DNA inserted between the first and second corresponding sequences of the nuclear DNA, resulting in replacement of the target sequence of DNA with the sequence of DNA of interest, wherein the target sequence has substantial similarity to SEQ. ID. NO. 1.